<About Firefly Luciferin>
In recent years, the visualization of biological events and phenomena have been considered to be important, and a demand has increased for an expansion of materials for visualization. At the same time, there is the demand for diversification of labeling techniques. In particular, labeling techniques for molecular imaging have been greatly developed in conjunction with progress in equipment of diagnoses and examinations. For example, labeling techniques that can be applied to cutting-edge technology, such as individualized medical care for cancer or heart disease, are being intensely researched. Along with progress in measurement techniques, the demand for higher-sensitivity and higher-performance equipment and labeling materials is quickly rising.
Among visualization techniques, a firefly bioluminescent system is considered to have an extremely high luminous efficiency and to be the system that most efficiently converts energy into light. Progress is also being made into deciphering the molecular mechanism of bioluminescence.
With a firefly bioluminescence, it is known that light is emitted due to a chemical reaction of luciferin, which is a luminescent substrate, by the action of luciferase which is the luminescent enzyme. In this reaction, the luminescent substrate is adenylylated (converted to AMP) within the luminescent enzyme in the presence of adenosine triphosphate (ATP) and divalent magnesium ions (Mg2+) and is derived into an adenylylated form, which is an active substrate. Next, this form is oxygenated, yielding a peroxide anion, and converted into dioxetanone, which is a high-energy peroxide. Unstable dioxetanone releases protons and carbon dioxide while decomposing and adopts an excited singlet state. The light emitted from this dianion-type excited singlet state is yellowish green, which is considered to be firefly light. The product after light emission is referred to as oxyluciferin.
As described above, the firefly bioluminescence has an extremely high luminous efficiency, and progress is being made into deciphering the molecular mechanism of bioluminescence. Therefore, a wide variety of luminescent material using a firefly bioluminescent system is being sold by many companies. In the development of luminescent material related to a firefly bioluminescence, however, the commercialization has mainly progressed in the field of medical biochemistry. Hence, while much research and development focuses in general on proteins (enzymes), very little research deals with low-molecular compounds (substrates). In particular, almost no correlation studies have been performed between an activity and a structure in which a luminescent substrate has undergone skeletal transformations.
Furthermore, even though luminescent enzymes can be supplied at low cost with a recombination technique, a luminescent labeling material using a firefly bioluminescent system supplied by a kit product or the like is expensive. The reason is that the luminescent substrate is luciferin. Currently, luciferin in D form, which is a natural luminescent substrate, is synthesized from D-cysteine, which is a non-natural amino acid. However, D-cysteine is extremely high in cost.
<Needs and Conditions of Long Wavelength Light Using a Bioluminescent System>
In order to measure a variety of phenomena, multicolor light emission is desired also for detection systems that use labeling. Therefore, the wavelength range of labeling material that can be used in a detection system is preferably wide. For example, in the research using multicolor light emission, labeling materials that emit light with a wavelength of approximately 450 nm to 650 nm or more as the label, particularly 680 nm or more, is preferably prepared. For in vivo labeling of deep portions, a red light emission labeling material is preferable since longer wavelength light yields better optical transmittance than shorter wavelength light. In particular, near-infrared light with a wavelength of 650 nm to 900 nm is used for the optical measurement of body tissue. Visible light (400 nm to 700 nm) is greatly absorbed by hemoglobin and other biological substances, whereas at wavelengths longer than near-infrared light, light is increasingly absorbed by water, so that light cannot proceed through the living organism. By contrast, the wavelength region of near-infrared light easily passes through the living organism and is therefore also referred to as a “window into the body”.
As described above, the firefly bioluminescence occurs by a chemical reaction between luciferin and luciferase. Using this fact, a luminescent enzyme may be created in advance by genetic engineering in a target organ, for example, and by subsequently dispersing luciferin throughout the body by intravenous administration or by intraperitoneal administration to the individual, the target organ that expresses the luminescent enzyme emits light. Furthermore, if cancer is transplanted to the target organ, the use is possible for visualization of cancer and for the basic research into regenerative medicine if the target organ is an organ from another organism. In particular, if the material is a long wavelength material that emits light in the region of the window into the body, the transmittance in the organism is high, and measurement from outside the organism is considered to be easier.
Currently, substrates with several emission wavelengths can be acquired as luminescent substrates for a firefly bioluminescent system. Examples of the shortest and longest wavelengths of the substrates include coelenterazine blue (approximately 480 nm) and firefly red (approximately 613 nm). Recently, longer wavelength red luminescent material (approximately 630 nm) using a railroad worm luminescent enzyme has become commercially available. Since longer wavelength light yields better optical transmittance, the latent demand is expected to exist for not only these emission wavelengths, but also for further expansion of the longest emission wavelength.
The following are examples of existing products that emit red and blue light using a bioluminescent system.                1. Promega KK: Chroma-Luc: approximately 613 nm (Non-patent Literature 1)        
This system uses a mutant click beetle and a native firefly luminescent substrate.                2. TOYOBO Co., Ltd.: MultiReporter Assay System-Tripluc: approximately 630 nm (Non-patent Literature 2)        
This system uses a red luminescent enzyme of the railroad worm and a native firefly luminescent substrate. The luminescent color is varied using luciferase genes for the colors of green luminescent luciferase (SLG, maximum emission wavelength of 550 nm), orange luminescent luciferase (SLO, 580 nm) and red luminescent luciferase (SLR, 630 nm). Luminescent enzymes yielding different luminescent colors are used.                3. University of Tokyo: Aminoluciferin: approximately 610 nm (Patent Literature 1)        
This discloses a luciferin derivative.                4. Promega KK: Chroma-Luc: approximately 480 nm (Non-patent Literature 3)        
This system uses coelenterazine and Renilla reniformis luciferase.                5. ATTO Corporation: Vargula hilgendorfii bioluminescence, approximately 460 nm (Non-patent Literature 4)        
This system uses a coelenterazine-based substrate and Vargula hilgendorfii luciferase.
The present inventors have also disclosed luciferin analog compounds in Patent Literature 2. These compounds have a similar skeleton to luciferin.
Furthermore, in Patent Literature 4, the present inventors have disclosed luciferin analog compounds that have a different skeleton from luciferin and that have a variety of emission wavelengths. For these luciferin analog compounds as well, there is a desire to shift the emission wavelength to an even longer wavelength.
Patent Literature 1: JP 2007-091695A
Patent Literature 2: WO 2007/116687A
Patent Literature 3: JP 2006-219381A
Patent Literature 4: JP 2010-215795A
Non-patent Literature 1: Promega KK General Catalogue 2008-9, 12.6
Non-patent Literature 2: Upload vol. 79, 2005 pp. 1-10, Toyobo Biochemicals for Lifescience 2006/2007 pp. 4-67
Non-patent Literature 3: Promega KK General Catalogue 2008-9, 12.14
Non-patent Literature 4: ATTO Corporation. General Catalogue 2008-2009, p. 247